Report for 9/8/2015 through 9/7/2016: New project: Adopting Emerging Reverse Genetic Tools. Crispr/Cas9 technology has made gene targeting in zebrafish straightforward and has opened the door to more elaborate genetic modifications, widely referred to as precise gene editing. Over the last year, Feldman has actively acquired, adapted and optimized reagents and protocols for Crispr/Cas9 gene disruption. This project has been greatly bolstered by the addition of a second Staff Scientist to the NICHD Zebrafish Core: Dr. Chon-Hwa Tsai-Morris to the NICHD Zebrafish Core. Dr. Tsai-Morris's main assignment has been to improve these genetic tools and generate mutants to NICHD Zebrafish Core customers as a new service. The Core has thus created and provided zebrafish carrying novel mutations in 15 distinct genes for the Weinstein, Sargent, Stratakis, Porter and Kaler labs. In the process of getting Crispr-Cas9 up and running, the Core targeted an additional 9 genes, for an overall success rate of >75% per locus-specific disruption construct, known as a gRNA. The core also established an optional quality-control strategy for selecting maximally mutagenized lines that will be particularly useful to zebrafish and Crispr/Cas9 newcomers. The above data were presented at the Spring 2016 Mid-Atlantic Regional Zebrafish Meeting, the Spring 2016 Mid-Atlantic Regional Society for Developmental Biology Meeting and the 2016 Allied Genetics Conference. Over 2015-16 Feldman engaged in research projects with six labs: four from NICHD, one from NCI and one from the Childrens National Medical Center. Porter Lab (NICHD): Zebrafish models of the human pediatric diseases. Smith-Lemli-Opitz Syndrome (SLOS) is an autosomal recessive, multiple malformation syndrome with pediatric onset characterized by intellectual disability and aberrant behavior. Phenotypic characterization is ongoing of zebrafish carrying mutant alleles of dhcr7, the zebrafish ortholog to the human SLOS gene DHCR7, which were generated with support from the Core in previous years. This project will continue in 2016-17. The Core also used Crispr-Cas9 technology to create three novel genetic mutant lines for the Porter lab. Phenotypic characterization by the Porter is ongoing and will continue into 2016-17. Stratakis Lab (NICHD): Function of zebrafish orthologs to human genes implicated in disorders of the pituitary-adrenal axis. (1) Gigantism arises due to excess growth hormone (GH) secretion during childhood, before the growth plates close. Since 2012, the Core has supported this labs investigation of the zebrafish ortholog to a human gene implicated as a driver of gigantism. The lab published a paper in June 2016, with Feldman as co-author, which included a description of this genes developmental expression in zebrafish. This year, the Stratakis lab also began to test the effect on growth and development of zebrafish in which this gene is chronically overexpressed in tissue-specific or ubiquitous locations, using the Gal4/UAS transgene system. These studies will continue in 2016-17. This year the Core used Crispr-Cas9 methods to generate for the Stratakis lab zebrafish carrying loss-of-function mutations in the above gene and three other zebrafish orthologs to genes implicated human growth anomalies. Characterization of the resulting phenotypes will begin in 2016-17. (2) Since 2012, the Core has also supported this labs investigation of the function of two zebrafish orthologs to human adrenal hyperplasia genes. Over previous years, the Core helped this lab generate and acquire, respectively, zebrafish carrying loss-of-function mutation for each of these orthologs. Phenotypic characterization has commenced this year with notable effects on juvenile growth in the case of one gene and on early embryogenesis in the case of the other. This project will continue into 2016-17. Kaler Lab (NICHD). Modeling copper deficiency-associated distal motoneuropathy The Menkes' gene encodes ATP7A, a copper-binding ATPase localized to the plasma membrane and the trans-Golgi network (TGN), which is critical for proper intracellular copper distribution. Two ATP7A missense mutations cause a milder syndrome than Menkes, a distal motoneuropathy that is nevertheless debilitating to children and young adults. Since 2013, the Core has supported a project to clarify the structure-function relationship of ATP7A and motor neuron defects from the perspective of these missense mutations. This project was awarded a Bench-to-Bedside grant, with $10,000 per annum funding to the NICHD Zebrafish Core through FY 2016-17. Over previous years the Core supported the Kaler labs work to visualize and compare motor neuron growth during embryogenesis of WT zebrafish embryos and embryos homozygous for null mutations in their ATP7A ortholog: atp7a. This comparison will continue into 2016-17. In parallel to these studies, Feldman and Tsai-Morris have made a concerted effort to establish genome-editing technology in the Core, using Crispr-Cas9 technology in combination with donor DNA and comparing two general approaches; double-stranded donor DNA for homologous-recombination-based genome editing and single-stranded DNA for homology-directed repair-based genome editing. The initial goal is to induce formation of zebrafish atp7a point mutations cognate to one of the human ATP7a motoneuropathy alleles. This work will continue into 2016-17. Tanner Lab (NCI). Assessing human metastatic cell behaviors in a whole-body (zebrafish embryo) microenvironment. The dual goal of this project is (1) to determine the trophic range of certain metastatic melanoma and breast carcinoma cell lines and (2) to document cellular dynamics during early tumor formation from metastatic cells that have seeded into new microenvironments. Feldman actively assisted the Tanner lab during the inception phase in 2013-14 and to a lesser degree in 2014-15. The Tanner lab worked very independently in 2015-16 and the Cores chief role has been to maintain and manage access to needed equipment, fish tank space and reagents. This level of very basic support is anticipated to continue into 2016-17. Tuchman Lab (Childrens National Medical Center). Finding neuroprotective drugs to mitigate hyperammonemia, a consequence of urea cycle defects & liver failure. Exposure of the brain to high ammonia causes neurcognitive deficits, intellectual disabilities, coma and death. Since 2012, the Core has helped this lab to use zebrafish embryos to identify small molecules able to diminish the effects of hyperammonemia. Over previous years, a library of hundreds of small molecules with known safety profiles for humans was screened and several promising candidates were identified for follow-up validation studies in zebrafish and other animal models. This year, the Core has helped the Tuchman lab increase throughput of this screen, bolstered by additional personnel from the Tuchman lab, and the Cores implementation of NICHDs massive embryo production systems (MEPS; see above) as a source for embryos.